Recombinant protein-based method for the delivery of silencer RNA to target the brain

ABSTRACT

The present invention relates to the design and development of recombinant protein for the delivery of silencer RNA complex to mediate RNA interference since it represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes across the blood-brain barrier (BBB). To overcome challenges due to this barrier for biologics and other biological complex, the present invention describes a method wherein peptide having sequence GGGGHLNILSTLWKYRC represented by SEQ ID NO. 1 known to target specific gangliosides was linked to a double-stranded RNA binding protein to bind and deliver silencer RNA to the brain parenchyma. The designed fusion protein comprising a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) and a brain targeting peptide sequence that binds GM1. Conformation-specific binding of TARBP2 domain to silencer RNA results in the formation of homogenous serum-stable complex with GM1 targeting potential. Uptake of the complex in neural cells reveals selective requirement of GM1 for entry. Remarkably, the invention pertains to the systemic delivery of the complex comprising TARBP-BTP and silencer RNA in AβPP-PS1 mouse model of Alzheimer&#39;s disease (AD) led to distinctive localization primarily in the cerebral hemisphere in the hippocampus and brain cortex and in principle can work across other mammalian CNS targets. Further, the delivery of silencer-RNA mediated by brain targeting peptide fusion led to significant knockdown of BACE1, a therapeutic protease target in both AβPP-PS1 and wild type C57BL/6 mice. The invention establishes the emergent importance of fusion proteins in delivering therapeutic siRNA as a simple complex to brain tissues to treat neurodegenerative diseases besides Alzheimer&#39;s disease (AD). The complex is also useful to study gene function of hitherto unidentified genes/interplay of genes in mammalian systems and central nervous system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/266,642 filed on Sep. 15, 2016, now U.S. Pat. No. 10,209,098, issuedFeb. 19, 2019. The entire contents of this application is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 18, 2019, isnamed 0147NF2016_US-Div Sequence Listing_ST25.txt and is 17,150 bytes insize.

FIELD OF THE INVENTION

The present invention provides methods useful for the delivery ofprotein-based complex containing silencer RNA to target cells or tissuesof the brain. The invention can be used to deliver pharmaceuticals tocross the blood-brain barrier in a non-invasive manner to delivernon-toxic biological complex to decrease levels of toxic substancesgenerated in subjects leading to neurodegenerative diseases.

BACKGROUND OF THE INVENTION

Short interfering RNAs (siRNA) as gene-specific therapeutic moleculesare resourceful tools to accurately control gene expression. However,delivery of these molecules to specific tissues is confronted due totheir anionic nature, large size and non-specific effects preventingtheir clinical utility. Additionally, the delivery of siRNA to the brainparenchyma is restricted by the blood-brain barrier hampering treatmentof subjects experiencing neurodegenerative conditions having long-termeffects. To overcome these limitations while exploring for selectivityand non-toxicity, many peptide carriers were earlier established byconjugating antibody ligands and fusion proteins to nanoparticlesthrough genetic engineering approaches, with the objective of targetingtransferrin and insulin receptors of endothelial cells lining the braincapillaries. In a prominent advancement, different from these studies,numerous cell-targeting peptides were chosen through phage display byvirtue of inherent tropism, increased avidity to mammalian cell surfacereceptors and ease of production. In an equivalent strategy, peptideswith the binding characteristics of tetanus toxin to trisialogangliosideGT1b led to the discovery of a 12-aa peptide Tet1, offering thepossibility of conjugating short peptides to larger protein scaffolds togenerate multi-functional fusion proteins with neuronal tropism.Succeeding these studies, in a similar effort, Georgieva et al.developed strategies to conjugate neurotropic peptides to lipid-basedmolecules to impart in vivo stability, which demonstrated remarkabletranscytotic capacity in vitro suggesting an identical mechanism invivo. Upon systemic delivery, the targeting molecules, having affinityfor GM1, were found to localize in the brain parenchyma and additionallyin the lungs mandating further explorations to understand the potentialof derivatized polymers displaying broad selectivity. Majority ofnon-viral vectors for nucleic acid delivery were earlier developed usingthe cationic lipids, cationic cell-penetrating peptides, and dendrimers.Spontaneous interaction of these molecules with nucleic acids led to theformation of stable non-covalent complexes. Knowledge-based rationaldesign strategies subsequently led to the usage of multiple componentsfor superior delivery and stability leading to successfultarget-specific gene silencing. Taking cues from neurotropic viruses,Kumar et al., fused the arginine peptide (9-mer) to a peptide derivedfrom rabies virus glycoprotein (RVG) to facilitate electrostaticinteraction with siRNA and specifically target acetylcholine receptors.The synthetic peptide fusion RVG-9R facilitated transvascular deliveryof siRNA resulting in target specific gene silencing. However, presenceof high density cationic charge on the carrier could lead to theformation of heterogeneous particles, non-specific biodistribution andlower yield of protein in suitable host systems.

RNA-recognition motifs conserved among double-stranded RNA-bindingproteins lend their attributes to the design of modular fusion proteins.In a study using an arginine-rich peptide, tandem repeats of TAT wasfused to the double-stranded RNA Binding Domain (DRBD) toelectrostatically bind siRNA. The approach, although facilitating thedelivery of siRNA into several primary cells including glioma lackedcell-specificity. It is thus evident that multiple DRBD motifs fusedwith cationic peptides may not confer additional in vivo advantage dueto their likely interaction with serum proteins, lack of targetselectivity and tendency to aggregate. Versatility of TARBP2 fusionprotein whose conformation-dependent binding to double-stranded RNAabolished the prerequisite of positively charged peptides. The strongbinding interactions led to the formation of neutral nanosized complexwhich were stable upon systemic administration. In the presentinvention, we have overcome the aforesaid barriers and invented a methodto deliver siRNA selectively to target the brain and central nervoussystem, by fusing the RNA-binding domain with a peptide having sequenceGGGGHLNILSTLWKYRC represented by SEQ ID NO. 9 that can retainflexibility and at the same time target GM1 and GT1b expressing cells,by virtue of their natural abundance of these receptors in neuronalcells to permit accumulation of the silencer complex with the aim oftargeting disease causing genes in neurodegenerative condition by theadvantage and ability of the peptide chimera to cross the blood-brainbarrier by receptor-mediated transcytosis by mediating efficient andeffective RNAi via GM1 in brain tissue.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to provide a method ofpreparation of a protein based complex comprising silencer RNA [siRNA]for target specific delivery to a ganglioside.

Another objective of the present invention is to provide a method fortarget specific delivery of a protein based complex comprising silencerRNA [siRNA] to a ganglioside.

Yet another objective of the present invention is to provide a proteinbased complex comprising silencer RNA [siRNA] for target specificdelivery to a ganglioside by receptor mediated transcytosis.

An objective of the present invention is to provide a method of treatingdiseases selected from the group consisting of alzheimer's, parkinsons,gliomas, and amyotrophic lateral sclerosis which comprises administeringto a subject an effective amount of protein based complex comprisingsilencer RNA [siRNA].

SUMMARY OF THE INVENTION

The present invention describes a method of preparation of a proteinbased complex comprising silencer RNA [siRNA] for target specificdelivery to a ganglioside, wherein said method comprises:

a) fusing RNA Binding domain [RBD] of human Trans Activation ResponseElement RNA Binding Protein [TARBP] with a brain targeting peptide [BTP]to form a fusion protein [TARBP-BTP];

b) cloning, overexpressing and purifying said fusion protein [TARBP-BTP]to obtain a purified fusion protein [TARBP-BTP]; and

c) associating said purified fusion protein [TARBP-BTP] with siRNA toform a protein based complex.

In an embodiment of the present invention, the ganglioside is selectedfrom GM1 or GT1b expressing cells.

In another embodiment of the present invention, RNA Binding domain isdouble stranded[dsRBD].

In a further embodiment of the present invention, TARBP-BTP protein andsilencer RNA is mixed in 5:1 mole ratio.

In an embodiment of the present invention, TARBP-BTP protein andsilencer RNA is mixed in 2.5:1 mole ratio.

In another embodiment of the present invention, silencer RNA is BACE1silencer RNA.

In a further embodiment of the present invention, BTP is having aminoacid sequence represented by SEQ ID NO. 9.

In an embodiment of the present invention, cloning is in pET28a plasmid,over expression in E. coli BL21(DE3) cells and purification using Ni-NTAaffinity chromatography.

The present invention also describes a method for target specificdelivery of a protein based complex comprising silencer RNA [siRNA] to aganglioside, wherein said method comprises the steps of:

a) fusing RNA Binding domain [RBD] of human Trans Activation ResponseElement RNA Binding Protein [TARBP] with a brain targeting peptide [BTP]to form a fusion protein [TARBP-BTP];

b) cloning, overexpressing and purifying said fusion protein [TARBP-BTP]to obtain a purified fusion protein [TARBP-BTP];

c) associating said purified fusion protein [TARBP-BTP] with siRNA toform a protein based complex; and

d) selectively targeting said protein based complex to the gangliosideby receptor mediated transcytosis.

In an embodiment of the present invention, ganglioside is selected fromGM1 or GT1b expressing cells.

In another embodiment of the present invention, RNA Binding domain isdouble stranded [dsRBD].

In a further embodiment of the present invention, TARBP-BTP protein andsilencer RNA is mixed in 5:1 mole ratio.

In an embodiment of the present invention, TARBP-BTP protein andsilencer RNA is mixed in 2.5:1 mole ratio.

In another embodiment of the present invention, silencer RNA is BACE1silencer RNA.

In a further embodiment of the present invention, BTP is having aminoacid sequence represented by SEQ ID NO. 9.

In an embodiment of the present invention, cloning is in pET28a plasmid,over expression in E. coli BL21(DE3) cells and purification using Ni-NTAaffinity chromatography.

The present invention describes a protein based complex comprisingsilencer RNA [siRNA] for target specific delivery to a ganglioside byreceptor mediated transcytosis, wherein said complex comprises RNABinding domain [RBD] of human Trans Activation Response Element RNABinding Protein [TARBP] along with a brain targeting peptide [BTP] andsilencer RNA.

The present invention also describes a method of treating diseasesselected from the group consisting of alzheimer's, parkinsons, gliomas,and amyotrophic lateral sclerosis which comprises administering to asubject an effective amount of protein based complex comprising silencerRNA [siRNA].

These and other features, aspects, and advantages of the present subjectmatter will become better understood with reference to the followingdescription and appended claims. This summary is provided to introduce aselection of concepts in a simplified form. This summary is not intendedto identify key features or essential features of the claimed subjectmatter, nor is it intended to be used to limit the scope of the claimedsubject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the designed plasmid construct encodingTARBP-BTP and the overall method to target the brain.

FIG. 2 is a cloning strategy by overlap PCR and DNA sequencing.

FIG. 3 (A) is a SDS gel electrophoresis followed by Western blotting ofpurified TARBP-BTP (13.378 kDa) and TARBP (without the targetingpeptide) 11.53 kDa. FIG. 3 (B) is a Matrix-assisted laser desorptionionization-time of flight mass spectrometry of TARBP-BTP. The major peakof the purified protein is 13.378 kDa.

FIG. 4 is a far-UV circular dichroism spectroscopy of TARBP-BTP.

FIG. 5 is depicting ability of complex to bind and protect silencer RNA(siRNA). Binding of the fusion protein to siRNA at 2.5:1 mole ratio withmaximal binding at 5:1 mole ratio (bottom gel). At these ratios, thecomplex was resistant to degradation by RNase A and this ratio was usedto prepare the functional complex for in vitro and in vivo experiments(top).

FIG. 6 depicts binding of TARBP-BTP complex with ganglioside GM1 whichis internalized by cells in vitro. 59.76%, 40.4% and 7.92% of cellsrespectively were found to be FAM-positive clearly indicating thatuptake is GM-1 dependent.

FIG. 7 depicts delivery of BACE1 silencer RNA in GM1-rich Neuro-2acells. In vitro functional knockdown mediated by TARBP-BTP led to 41%knockdown of BACE1 mRNA levels.

FIG. 8 depicts evaluation of cell viability. Non-toxicity of the complexTARBP-BTP:siRNA at all the indicated ratios in vitro.

FIG. 9 depicts biodistribution of the complex in vivo upon intravenousdelivery in AD mice. Localization of fluorescent complex is distinctlyvisible in the cerebral cortex and hippocampus (boxed area), whichclearly indicates delivery of the complex by transcytosis across theblood-brain barrier.

FIG. 10 depicts therapeutic delivery of AD-relevant silencer complex invivo in AD mice and C57BL/6 wild type mice mediated by TARBP-BTP:BACE1mRNA levels of the different brain regions and organs were measured byqRT-PCR. a) 8-10 week old wild type C57BL/6 mice 48 h after i.v.injection with 20 nmol TARBP-BTP complexed with 4 nmol BACE1 siRNA (5:1mole ratio) and compared with mice injected with complex containing 4nmol siSTABLE non-targeting control siRNA #1 or 4 nmol naked BACE1siRNA. b) BACE1 mRNA levels in organs of 8-10 week old wild type C57BL/6mice injected with TARBP-BTP:BACE1 siRNA. c) BACE1 mRNA levels in 10-12month AβPP-PS1 mice 48 h after i.v. injection with 20 nmol TARBP-BTPcomplexed with 4 nmol BACE1 siRNA (5:1 mole ratio). The values werenormalized to respective PBS control (100%). β-Actin served as aninternal control for all qRT-PCR experiments. d) Western blots ofrepresentative lysates from tissues obtained in (a and c) depict proteinlevels of the corresponding tissues. n=3 for each group. C=Control mice,T=Mice treated with TARBP-BTP:BACE1 siRNA complex. (* p<0.05 wereconsidered statistically significant when compared to PBS control group.NS=not significant).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1. pET28a Cloning vector (nucleic acid sequence)

SEQ ID NO: 2. TARBP-BTP nucleic acid sequence designed construct thatcan be expressed in Escherichia coli (nucleic acid sequence)

SEQ ID NO: 3. BTP Primer sequence: Synthetic primer designed for PCR(nucleic acid sequence)

SEQ ID NO: 4. Fg23 Primer sequence: Synthetic forward primer (nucleicacid sequence)

SEQ ID NO: 5. Rg 23 Primer sequence: Synthetic reverse primer (nucleicacid sequence)

SEQ ID NO: 6. BTP Primer sequence: synthetic primer for amplifying thebrain targeting ligand for expression in Escherichia coli (nucleic acidsequence)

SEQ ID NO: 7. FBTP Primer sequence: Synthetic primer for overlap PCR(nucleic acid sequence)

SEQ ID NO: 8. Rg-23 Primer sequence: Synthetic primer for PCR (nucleicacid sequence)

SEQ ID NO: 9. BTP amino acid sequence of the targeting ligand linked toTRBP2 that can be expressed in Escherichia coli (amino acid sequence)

SEQ ID NO: 10. Homo sapiens TARBP2, RISC loading complex RNA bindingsubunit (TARBP2), transcript variant 1, mRNA (nucleic acid sequence)

SEQ ID NO: 11. fTRBP2: Sequence corresponding to DNA originally isolatedfrom HeLa Cells from which RNA was isolated and used to synthesize cDNAby reverse transcriptase (nucleic acid sequence)

SEQ ID NO: 12. TARBP2 amino acid sequence: Translated amino acidsequence obtained from SEQ No: 11 (amino acid sequence)

SEQ ID NO: 13. Complete sequence of gene with Restriction site(TRBP2-CCCBTP): gene amplicon that can be generated in Escherichia coliafter cloning and transformation (nucleic acid sequence)

SEQ ID NO: 14. NdeI Restriction site (nucleic acid sequence)

SEQ ID NO: 15. XhoI Restriction site (nucleic acid sequence)

SEQ ID NO: 16: TARBP-BTP complete amino acid sequence: translated aminoacid sequence corresponding to the molecular weight of TARBP-BTP (aminoacid sequence)

SEQ ID NO: 17. TARBP-BTP nucleotide sequence: complete amplicon aftercloning and transformation of TARBP-BTP cloned in pET 28a and thentransformed in Escherichia coli (nucleic acid sequence)

DETAILED DESCRIPTION OF THE INVENTION

The invention reveals the groundwork of the functional formulation thatis a simple complex comprising of purified modular TARBP-BTP proteinchimera with the brain targeting ligand. The chimera is anendotoxin-free protein molecule having the ability to form a consistentand active complex with short silencer RNA bound in aconformation-specific manner. The resultant complex containing TARBP-BTPcontaining the silencer RNA selectively targets GM1. The targetingfunctionality fosters the ability to cross the blood-brain barrier andlocalize in the brain tissues to mediate therapeutic RNA interference(RNAi). The invention established in a mouse model reveals thatfollowing non-invasive delivery, the complex is capable of entering andlocalizing in brain tissues particularly the hippocampus and the cortexand to some extent the olfactory bulb and the striatum and decreaselevels of toxic peptide generation in these regions. These propertiesare the hallmark of a therapeutic pharmaceutical for treatingneurodegenerative condition additionally in other mammalian subjects.

EXAMPLES

The following examples are given by way of illustration of the presentinvention and should not be construed to limit the scope of presentdisclosure. It is to be understood that both the foregoing generaldescriptions and the following detailed description are exemplary andexplanatory only and are intended to provide further explanation of thesubject matter.

Example 1

Design, Cloning Strategy by Overlap PCR and DNA Sequencing

To construct recombinant TARBP-BTP fusion, gene sequence from TRAFcorresponding to the second domain (TARBP2/TRBP2) of the mammalianhomolog, a linker sequence encoding five glycine residues and DNAfragment encoding the brain targeting peptide sequence together with aC-terminal cysteine codon was amplified by overlapping PCR. DNA duplexeswith and without the targeting ligand were cloned in pET28a plasmid(Novagen) in the NdeI-XhoI site to generate N-terminal 6 His-taggedfusion constructs that were verified by DNA sequencing. TheN-terminus-tagged fusion protein TARBP-BTP, a 13.378 kDa with thecorresponding DNA and translated protein sequence by EXPASY. TARBPhaving high affinity to double-stranded RNA (dsRNA) was fused to aganglioside targeting peptide sequence, originally selected by phagedisplay for GT1b and GM1 binding to deliver siRNA to the brain.

Example 2

Purification and Verification of TARBP-BTP

The selected recombinants were expressed in E. coli BL21(DE3) cells andpurified to homogeneity using Ni-NTA affinity chromatography. E. coliBL21(DE3) cells overexpressing the recombinant proteins were lysed underdenaturing conditions using lysis buffer (50 mM sodium phosphate bufferpH 7.4 containing 300 mM NaCl, 10 mM Tris, 6 M urea and 1 mM PMSF)followed by sonication. Following centrifugation of the lysate at 18,000rpm for 20 min to pellet cell debris, the supernatant was incubated withthe pre-equilibrated Ni-NTA sepharose matrix for 1 h. The matrix wasthen loaded onto a column and washed with 0.1% Triton X-114 in lysisbuffer at 4° C. to remove the bacterial endotoxins. The matrix boundTARBP-BTP protein was refolded under native conditions by on-columnrefolding and eluted using sodium phosphate buffer (pH 7.4) containing300 mM imidazole. The eluted fractions were pooled, desalted and bufferexchanged in phosphate buffered saline (PBS) pH 7.4 using Sephadex G25superfine column and purity and quality validated by western blottingand mass spectrometry.

Example 3

Far UV CD Spectrum

Far UV CD spectrum (range 250-200 nm) of purified TARBP-BTP (0.1 mg/mlin PBS (pH 7.4) recorded at room temperature using JASCO-J-815spectropolarimeter equipped with Jasco Peltier-type temperaturecontroller (CDF426S/15). The secondary structure analysis of TARBP-BTPby CD spectroscopy reveals that the secondary structure of thesilencer-binding domain is unaltered. The spectrum, acquired in a 1 cmpath length cuvette, was an average of five scans that was corrected forthe buffer baseline and plotted using Origin? software (OriginLabCorp.).Spectra were recorded in ellipticity mode at a scan speed of 50 nm/min,response time of 2 s, bandwidth of 2 nm and data pitch of 0.2 nm. Meanresidual ellipticity was calculated as described. Further, far-UVcircular dichroism spectroscopy suggested a well-defined secondarystructure, consisting of both α-helices and β-pleated sheets similar toearlier observations.

Example 4

Ability of Complex to Bind and Protect Silencer RNA (siRNA)

The complex was prepared by incubating 20 pmol of siRNA to increasingconcentrations of TARB-BTP fusion protein in PBS buffer to obtain thepreferred mole ratios and incubated for 20 min prior to electrophoresis.For the protection assay, the complex with and without the targetingligand were incubated for 20 min followed by treatment with RNase A for1 h at 37° C. Samples were then extracted by phenol:chloroform:isoamylalcohol (25:24:1) and precipitated using ethanol as described previouslyand resolved by gel electrophoresis. The stability of TARBP-BTP andTARBP complex was further assessed by preparing the complex in PBS orDMEM media plus 10% serum followed by incubation for 1-6 h at 37° C.siRNA was then extracted by phenol-chloroform and resolved on 2% agarosegel and visualized by ethidium bromide staining. siRNA alone andTARBP-BTP or TARBP alone loaded in separate lanes serve as controls.Binding of the fusion protein to siRNA indicated strong association andformation of homogenous non-covalent complex that upon electrophoresisindicated strong binding and near-complete masking of siRNA at 2.5:1mole ratio with maximal binding at 5:1 mole ratio. At these ratios, thecomplex was resistant to degradation by RNase A and this ratio was usedto prepare the functional complex for the in vitro and in vivoexperiments. Out data reflects the ability of recombinant his-taggedTARBP-BTP and his-tagged TARBP protein to bind dsRNA in aconformation-specific manner, an attribute essential for in vivostability of the carrier upon delivery.

Example 5

TARBP-BTP Complex Binds Ganglioside GM1 and Internalized by Cells InVitro

The complex was prepared at 5:1 mole ratio, since maximal binding andprotection of siRNA was observed. Complex added to Neuro-2a, IMR32 andHepG2 cells in culture exhibiting varying levels of GM1 also depictedentry into cells via GM1 on the cell surface. This was supported in aFACS-based uptake assay evaluated in Neuro-2a, IMR32 and HepG2 cellsusing FAM-labeled siRNA complexed with TARBP-BTP. In such a condition,59.76%, 40.4% and 7.92% of cells respectively were found to beFAM-positive clearly indicating that uptake is GM-1 dependent.

Example 6

In Vitro Functional Knockdown of TARBP-BTP:siRNA Complex

Delivery of BACE1 silencer RNA in GM1-rich Neuro-2a cells. Knockdownassay using qRT-PCR in Neuro-2a cells, having the highest levels of GM1depicted that TARBP-BTP led to 41% knockdown of BACE1 mRNA levels.

Example 7

Non-Toxicity of the Complex TARBP-BTP:siRNA In Vitro

In the drawings accompanying the specification, the complex wasnon-toxic to cells when evaluated in an MTT cell-viability assay, whichindicated ˜90% viability at all the ratios examined. Cells were treatedwith TARBP-BTP:siRNA complex for 24 h and assessed in the presence andabsence of siRNA at the indicated mole ratios using MTT. Cells treatedwith PBS alone were considered as controls having 100% viability fromabsorption values measured using reduced formazan. Error bars indicatestandard deviation of triplicate sets.

Example 8

Biodistribution of the Complex In Vivo Upon Intravenous Delivery in ADMice

Firstly, for the preparation of the protein complex comprising ofsilencer RNA and TARBP-BTP fusion protein was prepared in 1×PBS pH 7.4.The purified protein solution is then filtered using 0.22 mm Milexfilters to get rid of any microbes and protein aggregates if any. Therequired amount of siRNA is also diluted in 1×PBS pH 7.4. Complexes areformed by mixing solutions of protein and siRNA in equal volumes eg. 100ml of protein and 100 ml of siRNA (the concentrations are adjusted insuch a way that the mole ratio is always kept at 5:1 protein:siRNA).Following the mixing, the solution is incubated at 4° C. for 15 min(incubation at RT may lead to the formation of aggregates). This complex(200 ml) is injected via the tail vein in mice using standard protocolsof delivery. In the drawings accompanying the specification, thedistribution of fluorescent complex (TARBP-BTP:siRNA) in AβPP-PS1 mousebrain upon intravenous delivery demonstrated its in vivo potential tocross the blood-brain barrier. This was executed by administeringfluorescent complex that were prepared by mixing corresponding amountsof fluorescent-TARBP-BTP and silencer RNA at 5:1 mole ratio. Miceinjected with the same volume of PBS served as negative controls. Micewere euthanized 6 h post-delivery of the complex and all the majororgans, including brain were dissected, sectioned and visualized. Eventhough AF⁶³³ signal originating from the labeled protein enabledcollection of fluorescent signals in the far-red spectrum with minimumbackground noise, non-specific fluorescence arising from all laser linesin the brain sections was eliminated by NaBH₄ and CuSO₄ treatment oftissue sections as described in the methods. In the representative brainsections of mice injected with Alexa Fluor⁶³³-TARBP-BTP:siRNA complex,the localization of fluorescent complex is distinctly visible in thecerebral cortex and the hippocampus region (boxed area), which clearlyindicates transcytosis of the complex across the blood-brain barrier. Toauthenticate that the observed signal arising from the 633 nm laser lineis from the labeled protein, the emission spectrum was matched with thatof AF⁶³³-TARBP-BTP fusion protein spotted separately on a coverslip,using lambda scan option in Leica SP8. In contrast, the lack offluorescence in the lung, liver and intestine and other organs such asthe heart and spleen indicated target specificity of TARBP-BTP.Fluorescence depicted in a different region of the brain i.e. braincortex, is shown in Representative sections stained with CD31 mark theendothelial cells of the brain capillaries. Significant Alexa Fluor⁶³³fluorescence originating from kidney tissue sections indicated excretionof the labeled complex through renal filtration.

Example 9

Therapeutic Delivery of AD-Relevant Silencer Complex In Vivo in AD Miceand C57BL/6 Wild Type Mice

The role of BACE1 in the cleavage of amyloid precursor protein (APP) andconsequent generation of Aβ peptide is known. Complex comprisingTARBP-BTP and BACE1 silencer RNA at 5:1 mole ratio was intravenouslydelivered. Both AβPP-PS1 and C57BL/6 mice were injected with the complexintravenously and evaluated 48 h post administration by qRT-PCR andwestern blotting. In addition to PBS control, naked BACE1 silencer RNAand TARBP-BTP complexed with non-targeting control silencer RNA werealso injected into C57BL/6 mice. FIG. 10 a-c demonstrates that a singledose delivered intravenously caused significant reduction in BACE1 mRNAlevels amounting to 35%, 47%, 38% and 48% in the cortex, hippocampus,prefrontal cortex and olfactory bulb respectively in C57BL/6 mice.Reduction in BACE1 mRNA levels in AβPP-PS1 mice were 35%, 43%, 24% and50% in the cortex, hippocampus, olfactory bulb and striatum respectively(p<0.05, except for hippocampus of AβPP-PS1 where p=0.078). The observedreduction in BACE1 mRNA levels evidently signifies the in vivo braintargeting potential of TARBP-BTP, an exceptional carrier of siRNA. Also,there were no changes in the BACE1 mRNA levels in liver, lung, kidneyand spleen. Two additional controls i.e. naked BACE1 silencer RNA andnon-targeting siRNA showed no significant changes in the BACE1 mRNAlevels, which indicated that the observed reduction in BACE1 mRNA levelsis significant.

ADVANTAGES OF THE INVENTION

In vivo experiments with BACE1 siRNA delivery mediated by TARBP-BTPclearly establish i) the in vivo stability, ii) tissue-specifictargeting across the blood-brain barrier and prominently iii) regionspecificity of TARBP-BTP:siRNA complex in successfully delivering BACE1validated silencer RNAs bound to the carrier complex. The functionalcomplex comprising the bi-functional chimeric peptide and BACE1 silencerRNA, a therapeutically relevant AD gene, would be capable of mediatingtherapeutic effects by the ability to cross the blood-brain barrier andenter the brain tissues particularly in sites of learning and memory toknockdown expression of the products responsible for generating toxicpeptides. The method is useful for targeting neurodegenerative diseases,e.g. AD and will find therapeutic applications in the central nervoussystem for other diseases.

Additionally, TARBP-BTP will be useful for functional analysis ofhitherto unknown genes. The delivery system due to conformation-specificbinding of the silencer offers a robust complex whose serum stabilityand controlled release in target tissues in a non-toxic andnon-immunogenic manner will find utility in clinical applications.

We claim:
 1. Protein based complex comprising silencer RNA (siRNA) fortarget specific delivery to a ganglioside by receptor mediatedtranscytosis, wherein said complex comprises RNA Binding domain (RBD) ofhuman Trans Activation Response Element RNA Binding Protein (TARBP)along with a brain targeting peptide (BTP) and silencer RNA; and whereinsaid complex is prepared by a method which comprises: a) fusing anucleic acid encoding a RNA binding domain (RBD) of human TransActivation Response Element RNA Binding Protein (TARBP) with a nucleicacid encoding a brain targeting peptide (BTP) having the amino acidsequence of SEQ ID NO: 9 to form a TARBP-BTP fusion nucleic acid fusionconstruct encoding a TARBP-BTP fusion protein having the amino acidsequence of SEQ ID NO: 16; b) cloning the TARBP-BTP fusion nucleic acidfusion construct into an expression vector, overexpressing and purifyingsaid TARBP-BTP fusion protein; and c) associating said purifiedTARBP-BTP fusion protein with an siRNA that targets the synthesis of aprotein to form the protein based complex.
 2. A method of treatingdiseases selected from the group consisting of Alzheimer's, Parkinson's,gliomas, and amyotrophic lateral sclerosis which comprises administeringto a subject an effective amount of protein based complex comprisingsilencer RNA (siRNA) according to claim 1.